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Characterization of triclosan-resistant mutants reveals multiple antimicrobial resistance mechanisms in Rhodospirillum rubrum S1H.

ABSTRACT: Antimicrobial resistance mechanisms were identified in 11 spontaneous high- and low-level triclosan resistance (Tcs(r)) mutants of Rhodospirillum rubrum S1H by genotyping complemented with transcriptional analyses, antibiotic resistance screening, and membrane permeability analyses. High-end Tcs(r) (MIC = 8 mg/liter) was the result of a FabI1(G98V) mutation. This point mutation led to an even higher level of Tcs(r) (MIC > or = 16 mg/liter) in combination with constitutive upregulation of mexB and mexF efflux pump homologs. Hence, a mechanistic synergy of constitutive efflux pump expression and a FabI1 point mutation could prevent TCS-induced cell permeabilization, which was shown to occur between 4 and 8 mg/liter TCS in the R. rubrum S1H parent strain. Low-level Tcs(r) mutants constitutively upregulated the emrAB, mexAB, and/or mexF homolog. The mutants that overexpressed emrAB also derepressed the micropollutant-upregulated factors mufA1 and mufM. In some cases, low-level Tcs(r) decreased innate resistance to ampicillin and tetracycline, while in others, a triclosan-induced antibiotic cross-resistance was shown for chloramphenicol and carbenicillin. This study showed that the TCS resistance degree is dependent of the initial exposure concentration in Rhodospirillum rubrum S1H and that similar resistance degrees can be the result of different defense mechanisms, which all have distinct antibiotic cross-resistance profiles.

SUBMITTER: Pycke BF

PROVIDER: S-EPMC2869150 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Characterization of triclosan-resistant mutants reveals multiple antimicrobial resistance mechanisms in Rhodospirillum rubrum S1H.

Pycke Benny F G BF Crabbé Aurélie A Verstraete Willy W Leys Natalie N

Applied and environmental microbiology 20100319 10

Antimicrobial resistance mechanisms were identified in 11 spontaneous high- and low-level triclosan resistance (Tcs(r)) mutants of Rhodospirillum rubrum S1H by genotyping complemented with transcriptional analyses, antibiotic resistance screening, and membrane permeability analyses. High-end Tcs(r) (MIC = 8 mg/liter) was the result of a FabI1(G98V) mutation. This point mutation led to an even higher level of Tcs(r) (MIC > or = 16 mg/liter) in combination with constitutive upregulation of mexB an ...[more]

PMID: 20305019

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Project description:Aiming at the development of a micropollutant biosensor in the frame of the Micro-Ecological Life Support System Alternative (MELiSSA), a pilot study was initiated to identify triclosan (TCS)-responsive biomarker genes in the MELiSSA carbon-mineralizing microorganism, Rhodospirillum rubrum S1H. TCS is a biocide, commonly found in human excrements and is considered an emerging pollutant in wastewater and the environment. Chronic exposure to MELiSSA-relevant concentrations (≥25 µg L-1) TCS caused a significant extension of the lag phase without affecting the growth rate. Analytical determination gave no indication of TCS biodegradation during the growth experiment and flow cytometric viability analyses revealed that TCS is only slightly toxic to R. rubrum. Through microarray analyses, the genetic mechanisms supporting the reversibility of TCS-induced inhibition were scrutinized. Hence, an extremely TCS-responsive cluster of four small adjacent genes was revealed, with up to 34-fold induction at 25 µg L-1. These four genes, for which the name micropollutant-upregulated factor (muf) was proposed; appear to be unique to R. rubrum and are shown for the first time to be involved in the response to stress. Moreover, numerous other systems that are associated with the proton-motive force were shown to be responsive to TCS, but never as highly upregulated as the muf genes. Hence, R. rubrum induced the phage shock protein operon (pspABC), numerous major facilitator efflux systems, cell envelope consolidation mechanisms, oxidative stress response, beta-oxidation, and carbonic anhydrase; while downregulating bacterial conjugation- and carboxysome synthesis genes. The muf genes and three efflux-related genes showed most potential as low-dose biomarkers. The two microarray experiments (10 and 25 µg l-1 Triclosan) were all performed in biological triplicate and containing three (in-slide) technical repeats. For all conditions, the Triclosan exposed sample (Cy5) was compared with the non-exposed solvent control (Cy3) to identify those genes that were differentially expressed upon Triclosan exposure.

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Characterization of triclosan-resistant mutants reveals multiple antimicrobial resistance mechanisms in Rhodospirillum rubrum S1H.

Publications

Characterization of triclosan-resistant mutants reveals multiple antimicrobial resistance mechanisms in Rhodospirillum rubrum S1H.

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